Published November 20, 1936
T H E EFFECTS OF CURRENT FLOW ON BIOELECTRIC
POTENTIAL
III. NIT~LLX
BY L. R. BLINKS
(From the Laboratories of The Rockefeller Institute for Medical Research, New York,
and the School of Biological Sciences, Stanford University)
(Accepted for publication, January 11, 1936)
a Blinks, L. R., Y. Gen. Physiol., 1929-30, 13, 495.
20sterhout, W. J. V., Ergebn. Physiol., 1933, 35, 967.
229
The Journal of General Physiology
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The effects of the flow of direct current on the bioelectric potential
of Nitella include: (a) polarization; (b) stimulation; (c) recovery or
restoration. Some of these have been described, directly or implicitly,
in a previous paper dealing with the direct current resistance of Nitetla
cells. 1
It was there noted that the high apparent l).e. resistance of the
protoplasm is accompanied by, and is probably largely due to, a
counter ~..~.F. built up by the current flow, which means that "the
previously existing bioelectrie potential has been altered to that extent.
Such regular counter E.~.F.'s will be designated "polarizations" for
convenience, without prejudice as to their real nature (i.e. whether
their time course corresponds to a static capacity or a polarization
capacity, although evidence points to the latter).
The profound changes of P.D. occurring during stimulation were
also briefly described,1 and have been the subject of many papers by
Osterhout and co-workers.2 The present emphasis will be upon the
current flow characteristics (threshold density, duration, direction,
etc.) which initiate stimulation, and the changes of polarizability
which occur during its course.
Finally, when the bioelectric potential has been greatly lowered,
and polarizability lost, either briefly during stimulation or more
permanently by treatment with KC1, current flows of proper direction
and density restore the normal P.D. and polarizability. These may be
called restorative effects.
Published November 20, 1936
230
~FFECTS O!~ CUI(R.ENT I~'LOW. III
T h e s e p h e n o m e n a will be s h o w n as c h a n g e s of the n o r m a l bioelectric
p o t e n t i a l r e c o r d e d c o n t i n u o u s l y f r o m string g a l v a n o m e t e r deflections
before, d u r i n g , a n d after c u r r e n t flows of d e s i g n a t e d value, m u c h as
w i t h Valonia 3 a n d ttalicystis, 4 as p u b l i s h e d previously. M a n y of the
p h e n o m e n a are c o m m o n to all these o r g a n i s m s a n d will be c o m p a r e d
as a basis for discussion of the m e c h a n i s m of c u r r e n t flow effects.
Methods
8 Blinks, L. R., Y. Gen. Physiol., 1935-36, 19, 633.
4 Blinks, L. R., J. Gen. Physiol., 1935-36, 19~ 867.
5 Blinks, L. R., J. Gen. Physiol., 1929-30, 13, 361.
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The electrical circuit was the modified Wheatstone bridge previously described. ~
This balances the purely ohmic resistances of the system, while allowing potentials
originating in the bridge (such as bioelectric potentials) to be recorded by the
vacuum-tube detector feeding into the string galvanometer.
Two differences of technique from the Valonla3 and Halicystis ~procedures may
be noted. (1) Owing to the extremely high resistances of the delicate glass capillaries necessary for insertion in Nitella cells, and the difficulty of maintaining a
supply of cells with these in place long enough for recovery to occur, impalement
was not attempted. Two external contacts of fixed dimensions (1 cm. long) were
employed, consisting either of agar blocks imbibed with the desired solutions, as in
the resistance measurements previously described, 1 or of solutions in the open ends
of small glass U-tubes, slightly notched to hold the cells. When, as in most cases,
it was desired to study the passage of current across only a single layer of protoplasm, one contact was treated with chloroform. This has been shown by Osterhout 2 to reduce the P.D. at that point approximately to zero, leaving that at the
other end to record alone, and usually unaltered, for some time (15 minutes or
more). While a shunting path around this potential source is offered by the cellulose wall, the latter is very thin and is imbibed with tap water or distilled water so
that its resistance between contacts is high-~ften 10 megohms or moreJ The
discharge of the bioelectric potential (100 to 200 my.) through this resistance
amounts to some 0.01 or 0.02 #a. (2) Aside from this leakage, which is unavoidable with external contacts, there remains the possibility of discharge of the bioelectric potential through the bridge itself, which is of course a completed circuit.
This was found of negligible importance in Valonia and ttalicystis, even through
the low resistances ordinarily employed with these cells; but in Nitella the danger
is more serious, since the discharge can itself easily become large enough to produce
stimulation. Two methods are at hand to control it: higher resistances in the
bridge, or compensation of the potential. The former was simpler and was accomplished by using as ratio arms radio "gridleaks" of 1 or 10 megohms each. With
the latter value the leakage through the bridge was reduced to the same magnitude
Published November 20, 1936
L. ~. BLINKS
231
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as that along the cell wall between contacts, making the total discharge, or residual
current, not over 0.02 to 0.04 ~a. Since the surface of the protoplasm at the standard contact of 1 cm. length was of the order of 0.1 to 0.2 cm. 2, the residual current
density was from 0.1 to 0.4 pa/cm. 2 of cell surface, averaging about 0.2 #a/cm. 2
This is usually insufficient to produce stimulation, although slight increments
upon this value may be sufficient to do so, as will be seen.
As a second method to reduce the residual current (that through the bridge)
compensation of cellular ~.M.F. was produced with an equal and opposite ~.M.F. in
series with the cell in its bridge arm. Sufficient records were taken in this way to
show that there were no significant differences due to the extra current flow in the
bridge, over and above those through the wall. However, since such records no
longer show the total P.D., but only its changes, they lose part of the advantage of
working across a single layer of protoplasm. I t is possible to correct this fault by
introducing a second, or "pseudobioelectric" potential in series with the detector in
the bridge diagonal. This "decompensates" the first compensating ~.M.g., without introducing a new current flow because it is in series with an electrostatic instrument drawing no current. But such an arrangement is cumbersome, necessitating frequent readjustment of both these ~.M.~.'S to follow changing bioelectric
potentials. If this adjustment is not made, current flow will be caused by the
compensating E.~r.F. at certain times, e.g. as when the cell's P.D. has been reduced
to a low or zero value during stimulation; this might be more disturbing than the
flow of residual current during normal conditions. All the records here published
were therefore taken without compensation of the cell's potential, just as with
Valonia and Halicystis.
Unless otherwise noted, records usually refer to the same cell throughout each
figure, and, with minor excisions to conserve space, are continuous.
The experimental current density values are marked in #a/cm. 2 below each
exposure on the records, an upward arrow signifying positive current passing inward across the protoplasm, a downward arrow, positive current passing outward.
These are of course the experimental increments or decrements effected on the
residual current as a base. They are furthermore the increments or decrements
which would have been produced if there were no counter E.M.~'. developed, i.e.
such as would have passed through the cell if dead or stimulated, or treated with
KC1 so that no counter E.~r.x~.appeared. They are therefore only transient initial
values which are instantly decreased as the counter E.M.F. develops, and their final
value in the steady state may be considerably less, e.g. a tenth or even a hundredth
of the original value, depending on the amount of counter ~.~.~'. developed. In
some cases, the outside ~.g.F. was not applied directly to the bridge, but through a
high external resistance in series with it (e.g. 10 megohms). This made charge
and discharge conditions more nearly alike, the charge occurring through this
resistance, the discharge through the ratio arms of equally high resistance. The
results with this circuit were not appreciably different, showing the time relations
to be determined more by the cell itself than by the external resistances, just as in
Valonia) This may be taken as evidence of polarization rather than static capacity.
Published November 20, 1936
232
EFFECTS OF C U R R E N T FLOW.
HI
80sterhout, W. J. V., J. Gen. Physiol., 1934-35,18~ 987.
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The bridge detector was the one-stage vacuum tube electrometer previously
employed. 5 I n its plate circuit was balanced a Cambridge portable string galvanometer, which gives essentially faithful recording of potential changes, its response being nearly linear over the field employed, and practically instantaneous
at the recording speeds used (about 1 cm. per second). The electrometer, working
at the free-grid potential of the vacuum tube (earlier a 201-A type, more recently
an 89 type, worked as ~ triode, for higher input resistance) drew practically no
current and served as an electrostatic instrument responding to the potential drop
along the part of the circuit which it tapped. In the bridge balanced with equal
ratio arms its sensitivity was therefore reduced to one half its open circuit value;
with the 10:1 ratio mostly used with Nitella, it had about 90 per cent of its open
circuit value.
Calibrations were introduced frequently on the records by an E.~.F. in series
either with the cell, or with the electrometer in the bridge diagonal. Calibrations
in the latter caused no flow of current, but had to be corrected by the sensitivity
factor (i.e. they were about 10 per cent higher than would be given by the same
potential in the position of the cells). Calibrations introduced in series with the
cell caused of course a change of current through the bridge, hence altered the bioelectric potential, in the usual curved course. True series calibrations were obtained, however, at the end of each run when the cell was chloroformed, and from
these are derived the millivolt ordinates inserted on each record. Here also the
value of the basal, ohmic resistance could be checked; this was often about 50,000
ohms and was balanced by 500,000 ohms in the resistance arm. The absence of
any deflection when currents were passed through dead cells (or a rectangular one
if the balance was not quite exact), indicates that the charge and discharge curves
obtained with living cells are really due to the protoplasm, and not to any spurious
capacities in the electrodes or bridge circuit.
The electrodes were non-polarizable, either Hg-calomel, or Ag-AgC1 frequently
recoated. Suitable salt bridges connected them to the cell contacts.
The source of experimental currents was a tapped battery of dry cells, from
which equal steps of voltage could be derived, the values applied to the bridge being
determined by a potential divider and voltmeter, as previously shown. 5 With 10
cells in the battery, as much as 15 volts could be applied to the bridge, giving,
through a dead cell of resistance 50,000 ohms, and a balancing resistance of 10
times this (due to the 10:1 ratio employed) a possible current of about 30/za, or a
maximum current density of 300/za/cm. 2 of cell surface. This is much larger than
is ever necessary to bring about the effects here described, for which a maximum of
about one-tenth this density is adequate in the most extreme cases (e.g. in the
presence of KC1). Usually not over 5 or 10 #a/cm. ~ was passed, but this is some 10
to 50 times the normal residual current density.
The temperature of the experiments ranged from 15 to 25°C., very few experiments being performed during the summer, when the cells are often difficult to
stimulate, s
Published November 20, 1936
L. R. BLINKS
233
7 Umrath, K., Protoplasma, 1932, 17,258.
8 Blinks, L. R., Rhodes, R. D., and McCallum, G. A., Proc. Nat. Acad. Sc.,
1935, 9.1,123.
9 Sen, B., Ann. Bot., 1931, 45, 527.
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A fall of effective resistance (disappearance of polarization), during the greatest
depression of P.D. in stimulation, was always found in the species of Nitella (mostly
N. flexilis) studied in New York with the technique described. This is in disagreement with the findings of Umrath ~ who reports that there is no change of effective
resistance during stimulation of the European species N. mucronata. Umrath's
results were obtained across a single layer of protoplasm by direct connection to
the interior of the cells (impalement). Current was pas~ed from an inserted
platinum electrode, while a pair of non-polarizable electrodes tapped the P.D.
Although polarization at the platinum electrode admittedly o~scured the results,
Umrath's Figs. 9, 10, and 11 show a clear increase of its current during stimulation. This increase is greater for larger currents, which would not be true if it
were due solely to the e.D. change. Umrath's concurrent P.D. records are difficult
to interpret since the purely ohmic IR drop of the system was not compensated.
Aside from these technical points, it is possible that the resistance of Umrath's
impaled cells was already low, so that the change during stimulation was not as
great as in the present cases. It was previously emphasized 1that Nitella cells must
be very carefully handled, to display their highest resistances. Impalement may
therefore introduce an injury which is only slowly recovered from. This is the case
in both Valonia ~ and Halicystis, 4 whose impaled cells for a long time display a
much lower polarizability than intact cells. Recent measurements on the California species Nitella clavata have shown also that the effective resistance even of
intact cells may sometimes be low, so that little change of polarization occurs on
stimulation, although the e.D. still alters in characteristic fashion. Umrath's cells
may have been like these. Polarizability and P.D. changes need not therefore
always go together, although they invariably did so in the records here presented,
and in hundreds of others like them. It is possible for example that polarization
occurs largely at one surface of the protoplasm, while the high e.D. originates at
another. The two could be altered simultaneously, or separately, somewhat as the
sources of e.n. in Halicystis may be individually affected, s
Another objection which might be fairly brought against the present technique
is that electrolytes could diffuse out from the cell during stimulation, and lower the
resistance of the shunting cell wall. While this is entirely possible, it would probably not occur as suddenly and completely as the observed changes, and be as
rapidly reversed again on recovery. In any case if the electrolytes came from the
cell sap, then the resistance of the protoplasm itself would probably be suddenly
lowered to release them. The experiments of Sen, 9 interpreted as showing such
diffusion of electrolytes out of Nitella cells during repeated stimulation, are ambiguous, for the cells themselves were included in the measuring circuit, and the observed resistance changes may have occurred in them, rather than in the adjacent
Published November 20, 1936
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FIG. 1
234
Published November 20, 1936
L. R. BLINKS
235
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FIG. 1. String galvanometer records showing charge and discharge curves of
counter E.M.F. resulting from the flow of direct current through an intact cell of
Nitella held between two aqueous contacts (tap-water, no chloroform). The
bridge is balanced to the effective D.C. resistance of the cell, here about 1,000,000
ohms (due mostly to the counter E.~.F.). Current densities are designated in microamperes per cm. 2 of cell surface (values at the beginning of current flow, before
counter E.M.F. develops). The duration of current flow is evident from the deflections at make and break; since the curve records the I'.D. of the right end of the
cell in relation to the left end, upward movements represent increased relative
negativity of the right end, downward ones increased positivity. The arrows indicate the direction of current flow through the cell; in Fig. la, the current flows from
right to left, and thus passes inward across the protoplasm at the right end, outward across that at the left. Increased positivity of the right end and decreased
positivity of the left result from this as the counter E.M.I~. develops.
I n Record a the first upward movement of the curve is due to the make of the
current: as the counter E.M.I~. builds up the curve comes back to the base line (of
resistance balance). Since the current at the right end is inward the counter
E.~.F. is outward, making the I'.D. more positive; at the left end the current is outward and reduces its positive P.D. When the current is broken the curve jumps
downward and then comes back to the base line (of no current flow) as the counter
E.~t.F. fades away. The curve at break is nearly a mirror image of that at make;
successive makes and breaks of larger currents merely increase the size of the deflections (there is an increasing unbalance of the bridge toward the end of Record
a) ; makes and breaks of current from left to right, in Record b, give similar curves
except for reversed directions; the left end is here becoming more positive and the
right end less positive as counter E.I~.F. develops. Up to about 3.0/za/cm. 2, only
such regular make and break curves appear (Records a and b). At this current
density, however, stimulation (s) occurs, on Record c, at the left end of the cell
(where current flow is outward). The positive 1,.D. of the protoplasm at that point
largely disappears, leaving that at the right end to record unopposed at break. I t
amounts here to some 120 inv. ; it disappears during the course of 15 seconds as the
positive P.D. is regained at the other end. Upon this altered 1,.D. as a new base,
current flows now produce different effects. At make there is a large deflection
away from the base, and the counter E.M.~'. developed is insufficient to bring the
circuitbacktobalance;theeffectiveresistance is lowered about 50per cent. As the
I'.D. recovers, and the base approaches zero, the counter E.~t.F. becomes larger and
restores the bridge balance. The counter E.M.F.'S in the early part of the curve
develop entirely at the right, unstimulated end of the cell; as the left end recovers,
they develop also there.
Record d shows stimulation at the opposite (right) end of the cell, due to passage
of current from left to right, hence outward at the right end. The effects are the
mirror image of those in c. I n both c and d, some polarizability (about 50 per cent)
remains, because only one end is stimulated, in each case, without propagation
down the cell. In e, f, and g, another, more sensitive cell is recorded, where stimu-
Published November 20, 1936
236
EFFECTS OF CURRENT FLOW.
III
Legend for Fig. l--Continued.--lation (sl) passes rapidly down the cell (s2) and both ends lose their P.D. and polarizability, to recover nearly in phase, so that the base line remains close to zero.
Here, however, nearly all counter E.~s.~. is absent for 2 or 3 seconds; at make of
current, the image deflects well away from zero, due to unbalance of the bridge, in
a nearly rectangular course, without curvature; then as recovery occurs, counter
E.M.~'. develops and causes the deflections to approach, and eventually reach, the
base line. In each make, the deflection is at first identical; thus in f the curve
shows that the deflection drops to the lower edge of the record each time the current is made, but lingers there less and less time as recovery occurs, back E.M.F.
developing and bringing the curve quite to the base line. This is difficult to see in
the latter part of the record because the curve rises so rapidly. This cell had a
lower threshold for stimulation in one direction than the other (as often occurs)
hence the first rectangular and later transient deflections away from base line in
Record g are about 2/3 as large as in f, corresponding to the current densities
employed (as marked).
On close inspection of the actual records, the momentary transient deflection
at make is seen to go to the same spot as in the rectangular deflections during
stimulation. A large current thus begins to flow at make, but is quickly reduced
by the developing counter E.M.F.
Sensitivity about 10 my. per horizontal division as marked. Time marks 1
second apart. Current densities as marked in #a/cm. 2, refer to the large transient
values at the instant of make, before decrease by counter E.~t.F. Repeated flows
of the same current density are designated in f and g by bars of proper duration.
10 Umrath, K., Proloplasma, 1932, 16, 631.
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film of water intended to receive the electrolytes. In addition Sen applied repeated and severe induction shocks, which, as Umrath 1° comments, may have
seriously injured or killed the cells, instead of merely stimulated them; without
concurrent P.I). measurements this cannot be ascertained.
More direct evidence is also available that such a mechanism cannot account
quantitatively for the observed changes in the present technique. A cell was
immersed for several minutes in 0.1 M NaC1, which does not stimulate the cells,
like KCI, yet has nearly the same conductivity as the vacuolar sap (about 10 per
cent less). During this time the salt must have diffused thoroughly into the wall.
The cell was then removed, drained, and measured in the usual way. While its
effective resistance was lowered, the decrease was only some 10 or 20 per cent (instead of 90 to 95 per cent occurring during stimulation l) showing that the crosssection of the cell wall is too small to offer such a low shunting resistance, even
when fully imbibed with a salt solution nearly like that of the sap. Polarizations
remained prompt and large, disappearing as usual only on stimulation, and reappearing on recovery. Unless, therefore, the electrolytes released during stimulation have a far higher specific conductivity than the cell sap itself, this explanation must be ruled out.
Published November 20, 1936
L. R. BLINKS
237
Experiments with Intact Cells
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Very regular curves characterize the building up of counter E.•.F.
during current flow, and its disappearance on the break of current.
A few examples of these were included in a paper on the resistance of
Nitella. 1 Fig. 1 gives a wider range of the same sort taken with both
contacts uninjured (tap water; no chloroform), and with the bridge
balanced to the steady state, or effective resistance, here about 1 megohm. The curves therefore deflect sharply away from zero at make
of current and approach the zero line as the counter ]~.1~.]~. builds up,
to remain there as long as the current flows (steady state). At break
they deflect again to the same extent as at make, but in the opposite
direction, approaching the zero line in a regular curve as the counter
]~.~.F. disappears. Deflections resulting from 9 or 10 equal increments of current are shown, first in one direction through the cell, and
then reversed. A marked regularity and similarity of charge and discharge curves, during successive exposures, and with different current
densities, may be noted.
Finally, however, at sufficiently high current densities, a stimulation
(S) occurs (at the contact where the current passes outward across the
protoplasm). Not only is the P.D. altered, giving the characteristic
"negative variation", but the effects of current flow also become quite
different. With the bridge maintained at the original balance (1
megohm), the "charge" curve no longer fully approaches the base line
(now the altered P.D. of the action current) during current flow, but
flattens out somewhat above or below it, depending on the direction
of current flow, and only tends to come back fully as the P.D. itself
recovers. Still more strikingly, if the stimulation is not confined to
its point of origin (cathode) but succeeds in passing down the cell to
the other contact, there is for a moment no counter ~..M.]~. whatever
developed during current flow, as shown in Fig. lf. The deflection is
abruptly rectangular, with no curve at make or break, showing that
the resistance of the cell is purely ohmic, and much lower than the
high resistance employed to balance the effective resistance of the unstimulated cell. Then as the cell recovers from the stimulus at both
contacts, the deflections become more curved, approach closer and
closer to the (balanced) base line during current flow, and eventually
reach it again.
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238
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d
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L. R. BLINKS
239
FIG. 2. Records showing charge and discharge curves of counter E.~t.l~. developed by flow of direct current through an intact cell of Nitella, much as in
Fig. 1 except that the bridge is now balanced to the true or ohmic resistance value
(here about 50,000 ohms, largely due to the vacuolar sap). The counter E.•.F.
therefore builds up to its full value above or below the zero line, depending on the
direction of current flow, and is nearly proportional to current density, up to stimulation values, when it is decreased, to build up again on recovery. (here quicker than
in Fig. 1).
Current densities in ~a/cm. 2, upward arrows signifying passage of current from
right to left, downward arrows from left to right. The potential is that of the right
end, which becomes positive to the left during the flow of inward current, negative
during outward flow, and stimulates at a threshold of 0.8 to 1.0 ~a/cm. 2 (one stimulation occurs at 0.3/za/cm. 2 in Record a). The bars in c and d signify the duration
of repeated flows of current of the same density as the last previously marked.
Sensitivity about 10 my. per horizontal division, as indicated. Time marks 1
second apart. No residual current (except during stimulation), both ends being
intact.
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Records of somewhat different a p p e a r a n c e b u t of essentially the
same sort are obtained if the bridge is balanced, not to the effective
resistance (which includes polarization) b u t to the true or ohmic resistance alone, which is largely t h a t of the vacuolar sap between the contacts. Fig. 2 shows records of this sort, still using an intact cell with
b o t h contacts normal. I n s t e a d of starting as a sharp deflection a w a y
from zero, and then approaching it as the counter E.M.F. builds up, the
l a t t e r now starts at zero, and reaches a value above or below this
base line corresponding to the direction and density of the current.
T h e curves, as regular and s y m m e t r i c a l as in Fig. l, are merely m o v e d
up or down in the field. T h e y show more clearly the p r o p o r t i o n a l i t y
of counter E.M.F. to current density, up to the threshold for stimulation. Here again the base line becomes distorted, due to loss of P.u.
at one contact, and during this time the polarizations become of smaller
magnitude; if the stimulation spreads to the other contact t h e y m a y
become m o m e n t a r i l y suppressed. Since the bridge is balanced to the
ohmic resistance of the cell, the loss of polarizability is shown b y the
decrease (or loss) of all deflection; e.g., Fig. 2c. Polarizability reappears as the cells recover from stimulation; in the figure it appears to
be slower t h a n the recovery of P.D., b u t this results from recording
two intact regions, whose opposing potentials recover nearly in phase
and so cancel each other. Pictures truer in this regard, as well as in
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240
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L. R. BLINKS
241
showing o t h e r directional effects w i t h o u t confusion, are o b t a i n e d b y
r e c o r d i n g o n l y one p r o t o p l a s m i c P.D., the o t h e r h a v i n g b e e n d e s t r o y e d
b y chloroform.
Experiments with One Contact Chloroformed
FIG. 3. Records showing directional effects across one intact layer of protoplasm
in Nitella. The zero has been moved to near the top of the records. Chloroform
is then applied (CHC13, Record a) at the left end of the cell, abolishing its potential
and allowing that at the right to record in its entirety (about 120 my.). The cusp
(c) may be noted during this process; it corresponds closely to that occurring during
stimulation (s). A current now passes continuously due to discharge of the bioelectric potential through the bridge circuit; this residual current is about 0.2
/za/cm. 2 at the beginning and falls to about 0.13/~a/cm. 2 in Record e.
Record a shows the result of causing an inward current to enter at the intact
region (right end) : counter E.M.F. develops nearly proportionally to current density,
raising the total I'.D. to 150 my. Record b shows outward currents (superimposed
upon the residual current) ; these produce polarizations also, but at a threshold of
0.4/za/cm. 2 (total 0.6/~a), a stimulation occurs, the 1,.D. drops to about 30 my., and
polarization entirely disappears, slowly reappearing. No further stimulation
occurs at this density, but at 0.6/za superimposed, in Record c, another stimulation
occurs with the same characteristics, d and e show further increases of threshold,
with finally a second stimulation occurring at 1.0/*a in e.
Current densities in/za/cm. 2 as marked, bars indicating the duration of repeated
current flows. Sensitivity about 8 inv. per horizontal division, with calibrations
as marked (a series calibration in c shows polarization charge and discharge due to
current flow in the cell). Time marks, 1 second apart.
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Fig. 3 shows the characteristic c h a n g e which occurs w h e n chlorof o r m has been applied to one end of the cell; owing to the disappearance of p o t e n t i a l at this point, the recorded 1,.D. rises f r o m zero ( n o w
m o v e d near the t o p of the record), to the full positive values of 100 t o
200 m v . w h i c h characterize the p o t e n t i a l across the p r o t o p l e s m of
Nitella. T h e cusp (C) is to be n o t e d ; it is a l m o s t i n v a r i a b l y f o u n d
b o t h during the chloroform injury, a n d the action c u r r e n t or n e g a t i v e
variation.
C u r r e n t is n o w passed, with the bridge b a l a n c e d as before to the
o h m i c resistance of the cell. T h e same regular reproducible curves
are f o u n d as before, b u t these n o w show definitely as increases or
decreases of the original bioelectric potential, a n d it is possible to dis-
Published November 20, 1936
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242
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L. R. BLINKS
243
Valonia2)
Another effect of large current density, stimulation at the break of
inward current flow, is best discussed after consideration of outward
currents, since it is probably to be referred to the same mechanism.
Outward Currents.--These decrease the existing 1,.n. in regular curves
comparable to the increases produced b y inward currents. Examples
are shown in Figs. 3 and 4. The magnitude of change is again proporFIG. 4. Records showing characteristics of stimulation and polarization, much
like those of Fig. 3, except that the 1,.D.here goes to zero or slightly negative during
stimulation. One end of the cell has been previously chloroformed, incompletely,
at first, leaving a small 1,.n. which is responsible for the apparent negativity at
the other end when stimulated. The threshold, at first 0.6 #a/cm. 2 (and here
showing a long delayed response) rises to 1.8, 2.7, and ~%allyto 3.0/~a/cm.2(Record
d), where a second stimulation occurs rather promptly. Finally, after about 1
minute between Records d and e, stimulation occurs again at 1.5/za/cm. 2
Note the disappearance of polarization during the maximum depression of I'.D.
and its reappearance during recovery.
Currents are as marked in #a/cm. 2, all outward across the intact protoplasm
(downward arrows) except four flows of inward current in c (upward arrows). Sensitivity about 8 my. per horizontal division, with values as marked. Time marks
1 second apart. Duration of currents indicated by bars.
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tinguish between the effects of currents passing inward and outward
across the protoplasm. These m a y be taken u p separately.
Inward Currents.--The counter E.~.F., opposing the flow of such
current, increases the existing potential, making it still more positive.
The a m o u n t of increase is nearly proportional to the current density
up to fairly high values; sometimes to some 8 or i0 # a / c m . 2 of cell
surface although usually only up to 4 or 5 # a / c m . 2 The 1,.9. m a y be
increased thereby as much as 100 or 200 Inv. above its normal value,
or to a total of some 300 or 400 Inv. (This enhanced value is apparently the highest bioelectric potential yet measured in a single cell.
T h a t in Halicystis seldom exceeds 100 Inv., positive or negative; 4 and
the small negative t,.D. of Valonia m a y be reversed to 200, or at most
300 Inv. positive b y the flow of current inward2) This appears to be
an upper limit above which the P.o. cannot be further increased, even
by much higher currents; proportionality of deflections falls off above
this, with the characteristic cusps and recessions shown in Figs. 6 and
7. (Much the same situation holds for large inward current flows in
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FIG. 5
244
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L. I1. BLINKS
245
FIG. 5. Records showing stimulation and recovery in Nitella, with particular
reference to the increase of threshold, and the effect of inward current flow in
speeding recovery. The cell has been previously chloroformed at one end, so that
the P.D. is that across one contact. This is proportionally increased, in regular
counter E.M.F.'s to about 150 my. by the flow of positive current inward in Record
a. Prompt stimulation then occurs with 1/za/cm3 outward flow in b; succeeding
flows of this current produce at first no polarization (rectangular deflections being
due to an ohmic unbalance (corrected at the arrow in d), then an increase. The
threshold rises to 1.2 #a/cmJ in c, 1.4 in d and e, and 1.6 in/. In the latter record
the current is reversed from outward to inward at the arrow, and with it the direction of counter E.~.F. Note here, that the recovery of positive P.D. is somewhat
faster than in the preceding Record e where successive outward flows were passed.
Sensitivity about 8 my. per horizontal division, as marked, and as shown by the
50 mv. calibration in c (in series with the cell, therefore producing current flow and
polarization with a curved course). Time marks 1 second apart. Bars indicate
duration of current flows of the density previously marked.
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tional to current density over a limited range, b u t the proportionality
ceases at a much lower value than with inward currents, and with an
opposite effect, since a much larger, instead of smaller, change of P.D.
then occurs ("stimulation"). The extent of proportional P.D. change
varies from cell to cell, b u t is seldom over 50 or 60 my. (produced b y a
current of not over 0.5 or 1 # a / c m 3 ) . With larger outward currents,
the curve inflects, instead of flattening out to a steady value, and the
potential falls away to low values (as in Fig. 3) or to zero.
This curve is m u c h like that produced b y the flow of outward current
upon the potential of Halicystis, 4 which also has a sigmoid course, and
a cusp near the apex. I t differs, however, in the following respects:
(a) The P.D. of Nitella seldom actually reverses in sign during
stimulation; this m a y only mean, however, t h a t in Halicystis there
is a second (negative) source of potential (e.g., at the inner surface of
the protoplasm) which is not affected b y current flow, while in Nitella
this either does not exist or is affected b y current flow nearly simultaneously with the positive source.
(b) I n normal Halicystis a continued flow of current is necessary to
carry the effects to completion, and to maintain reversed r.9. Stimulation in Nitella, once initiated b e y o n d a certain point b y the threshold
current density, goes on to completion even if the current is stopped.
This is shown in Figs. 3, 4, and 5 where the current is interrupted at
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246
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L. It. BLINKS
247
various points along the curve: This is more like the situation in
which has been t r e a t e d with a m m o n i a in concentrations
close to the critical reversal value. 4
(c) R e c o v e r y is also largely independent of the flow of current in
Nitella, often occurring even though the original stimulating current
continues to flow. I n Halicystis the P.D. remains reversed as long as
the exciting current flows (or even if it is considerably decreased in
value) positivity being regained only with m u c h lower or zero o u t w a r d
currents.
T h e m o s t striking change occurring during stimulation is the disa p p e a r a n c e of counter ~.M.F. noted before with intact cells. Figs: 3,
4, and 5 show t h a t as the ~.3. reaches its lowest value, just after the
cusp, renewed passage of o u t w a r d current produces little or no deflection a w a y from the base line, although the same current h a d just previously produced a typical " c h a r g e " curve which preceded and passed
over into stimulation. This absence of polarizability continues to
hold for a second or two, then as the P.9. begins to build u p again,
charge and discharge curves also reappear, becoming larger as r e c o v e r y
Halicystis
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FIG. 6. Records showing polarization and stimulation in Nitella, largely b) inward currents, with special reference to stimulation at break of the latter. The
cell has been previously chloroformed at one contact so that the record is that of
the I'.D. across the protoplasm at one contact only. Small inward and outward
currents produce very regular polarizations in a. At the break of 0.75 #a/cm. 2
inward, a very slight overshooting is evident; this becomes more pronounced at
break of 1.0/~a/cm. 2 and passes over into characteristic stimulation at break of
1.25/~a/cm. 2 Succeeding currents of this density elicit at first small then increasing polarizations, with finally an incomplete stimulation at the end of a, (as shown
by the lessened polarization in b). In b, at the increased threshold of 1.5 #a/cm. 2
true stimulation again takes place on break of inward flow. Increasing polarizations mark recovery, but the threshold is now raised to 2.25 #a/cm. 2 in Record c,
where a break of 2.5/~a/cm3 finally causes a quick, rather incomplete stimulation.
Outward currents are now passed again in d, resulting in slight stimulation at
0.75 ~a/cm. 2 and again at 2.0 #a/cm3, but otherwise only in cusped polarizations,
with overshooting on the break; these occur both with inward and outward currents
in Record e.
Sensitivity about 8 inv. per horizontal division, as indicated. Time marks 1
second apart. Bars indicate repeated flows Of current of the density previously
marked.
Published November 20, 1936
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Published November 20, 1936
L. R. BLINKS
249
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proceeds, and eventually reach their original size and shape. Polarizability to inward currents is recovered pari passu, as shown in Fig. 5f.
Polarizability and a large bioelectric potential are thus closely
parallel in time, and may even have the same structural or metabolic
basis. This recalls the situation in Valonia, where very slight polarizability is often displayed by cells in their normal low and slightly
negative P.D. level, while if this is made positive by acid sea water,
cresol, or the flow of inward current itself, more regular polarizations
also appear. 3
A further interesting behavior of Nitella is also shown in Figs. 3, 4,
and 5 where a succession of identical currents were passed during and
after stimulation. It is seen that a second stimulation does not occur
with the same current density which produced the first, even some 30
seconds to a minute after recovery seems complete (i.e., after the P.D.
has reached a constant high value). Renewed stimulation occurs only
when a slightly larger outward current is passed, and this process may
be repeated several times with increasing current density. This
appears to be a real increase of the threshold for stimulation; for,
although the P.D. itself may recover to a slightly lower positive value
each time, thereby decreasing the residual outward current which is
additive to the imposed currents in producing stimulation, this reduction of residual current is not usually as great as the rise of additional
current necessary. It appears instead that the P.I). must be driven to
successively lower levels (i.e., larger polarizations produced) by outward currents before stimulation occurs. Only at rather high current
density (1.0 #a/cm> in Fig. 3, 3.0 /~a/cm. ~ in Fig. 4), does a second
stimulation occur within 20 or 30 seconds.
It is conceivable that the increased threshold for stimulation after
recovery is due to the same compensatory mechanism which causes
recovery itself (even during continued outward current flow). There
might be produced a substance (e.g. an acid) which only slowly diffuses away or reacts, hence accumulates and requires a progressively
larger current to counteract its effects. This period of enhanced
threshold may be called a relative refractory period. There is also an
absolute refractory period, covering the greatest depression of P.D.,
but also extending into part of the recovery phase, when even extremely large outward currents do not produce typical stimulation,
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Fro. 8
250
Published November 20, 1936
Lo R. BLINKS
251
only an irregular polarization, with cusps (Figs. 6 and 7). This m a y
occur more and more as the result of repeated stimulation (Fig. 6), and
probably is correlated with the low positive P.I~. reached in this condition, so t h a t action currents of normal magnitude become impossible.
I t is therefore to be distinguished from those cells reported by Osterhour and Hill, 6 which are either naturally or experimentally not capable of stimulation, yet which m a y be rendered sensitive by treatment.
(Such cells have a high positive p.I).)
In contrast to the behavior of nerve, there appears to be no hypersensitive period in Nitella during which excitability is heightened,
following an action current.
Special Effects of Inward Currents
FIG. 8. Records showing polarization and partial stimulation by current flow
in Nitella. Particularly are to be noted the quick spike-like response, either to
break, of inward current or during outward flow, followed by rapid recovery, a
second upward movement and recovery, and even a third, the series dying away in
decremental fashion, like a damped oscillation. NaC1 solutions tend to produce
this effect.
Currents in/~a/cm?, direction being indicated by arrows, inward current upward
and outward current downward. Sensitivity about 7 my. per horizontal division,
the P.D. being that of one contact only (the other contact chloroformed). Time
marks 1 second apart.
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These include, in addition to the regular polarizations considered
above, stimulation at break, and restoration of positive potentials.
Break E~ects.--It was mentioned above t h a t at the break of inward
currents, stimulation sometimes occurred. Examples are shown in
Fig. 6. There is nothing startling about this, which should be expected as a result of a temporarily greater residual outward current,
due to discharge of the heightened positive potential produced by
inward current. I t would be expected in those cells with the lowest
threshold for stimulation by outward currents, and this was generally
found to be true. Fig. 6 also shows again t h a t recovery of polarizability after stimulation occurs for inward as well as outward currents;
indeed it m a y be aided by such current flows. Before taking up this
restorative function, two other points m a y be noted. One is t h a t a
second stimulation does not usually follow on breaks of successive
Published November 20, 1936
252
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~'~
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L. R. BLINKS
253
FIG. 9. Effects of current flow on Nitella cell exposed to KC1. The cell was
previously chloroformed at one contact; 0.05 • KC1 was then applied at the intact
end, giving the decrease (and slight reversal) of P.D. shown in Record a. Currents
were then passed as shown, densities up to 15/~a/cm. 2producing only slight counter
E.M.~. At 15 #a/cm. ~ inward current, however, a large polarization results, with
sigmoid course and a marked rise to a sharp cusp, followed by a recession. On
cessation of current flow, the counter E.M.F. drops away abruptly. Larger densities now produce more rapid polarizations, which, however, do not rise to appreciably higher values and do not show the cusp. After these flows a return to 15
/~a/cm. 2 is ineffective; only at 25/~a/cm. 2 does a good polarization again appear,
and here again a third flow is ineffective (Record c). These curious alternations of
response are probably due to breakdown after the cusps, which requires some time
for repair; in fact finally, as in Record e, the polarization may entirely die away
during the flow of current.
Current densities in /~a/cm. 2 as marked, inward flows being designated by
upward arrows, outward current (only once, in a) by a downward arrow. Sensitivity about 8 my. per horizontal division as marked on each record, and shown by
calibrations of 50 my. in Records a and b. Time marks 1 second apart.
11Osterhout, W. J. V., and Hill, S. E., J. Gen. Physiol., 1934-35, 18, 499.
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later inward currents, of the density originally effective (Fig. 6), showing an increase of threshold analogous to t h a t for o u t w a r d currents.
This does not always hold, however, as shown in Fig. 7, where it
appears t h a t large o u t w a r d flows m a y sensitize the cell to b r e a k stimulations to a greater degree t h a n to normal o u t w a r d flow. A second
point is t h a t stimulation at b r e a k m a y be incomplete, just as at m a k e ,
the depolarization curve beginning to " o v e r s h o o t " b e y o n d the original
P.D. line, b u t recovering without complete stimulation. E x a m p l e s are
shown in Fig. 6. This is p r o b a b l y due to too short a flow of the augm e n t e d residual current, which falls to a subthreshold value before
stimulation is thoroughly initiated.
Sometimes a second or even a third partial stimulation of this sort
occurs, either during outward flow, or at the b r e a k of inward current,
m a k i n g a decremental ("die-away") series as shown in Fig. 8. T h e r e
is no fully satisfactory explanation of these " d a m p e d oscillations"
which p r o b a b l y represent the interaction of the stimulated and n e a r b y
regions, without a true action current occurring. Osterhout has
recently suggested t h a t they represent effects at only one (probably
the inner) surface, the circuit being completed through the protoplasmic interior without the participation of the other (outer) surfaceY
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254
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L. ~. BLINKS
255
FIG. 10. Records showing further characteristics of response to inward current
under KC1 treatment. The Nitella cell has previously been chloroformed at one
end and exposed to 0.05 • KC1 at the recorded end. Inward current is then
passed. Small though regular polarizations are evoked by currents up to 8/za/cm. 2
At 10/~a/cm. 2 a sigmoid polarization produces over 200 my. positive P.D. A repetition of this produces a slightly faster response, while higher currents greatly
accelerate the time course which becomes very abrupt at 20 /~a/cm. 2, without, however, greatly increasing the positive •.B. reached. Record b shows a
second series of inward currents, at first increasing in density, then decreasing.
c is a record of another cell, similarly treated; and d and e of a third cell, showing
the general similarity of responses. In e, note particularly that the response to
successive flows of 12.5 #a/cm. 2 becomes somewhat faster; and that to higher densities much faster, but the positive P.3. reached is somewhat less, apparently due to
an injury following rapidly on the sharp cusp.
Sensitivity about 13 mv. per horizontal division (50 my. calibration in d).
Time marks 1 second apart. Current densities in/~a/cm, z, all inward (as indicated by upward arrows).
The resemblance of these curves to those taken with Valonia, previously published 3, is striking.
12Blinks, L. R., Proc. Soc. Exp. Biol. and Med., 1932-33, 30, 756.
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Somewhat analogous r h y t h m i c effects h a v e been f o u n d in Halicystis,
so far unpublished; there t h e y suggest a delayed response (possibly
metabolic) to the stimulus, resulting in t e m p o r a r y overcompensation,
followed b y recovery, then a renewed stimulation, etc.
Restorative Effects.--It was m e n t i o n e d a b o v e t h a t recovery of P.n.
after stimulation m a y be speeded up b y a continued or even an interrupted, flow of current inward across the p r o t o p l a s m . E x a m p l e s
suggesting this are included in Fig. 5, although the normal recovery
varies sufficiently in speed to m a k e it s o m e w h a t uncertain.
More striking evidence of the restorative action of inward currents
comes from experiments with cells which h a v e been exposed to a
threshold concentration of KC1 (0.01 ~ or higher). Reasons h a v e
been previously given 12 for regarding such cells as p e r m a n e n t l y stimulated, since the KC1 appears to produce its m o r e profound effects
largely b y inhibiting recovery from stimulation (whether induced
electrically, mechanically, or, as often happens, b y the K C I itself,
either i m m e d i a t e l y on application, or after a delay). T h e P.v. now
remains p e r m a n e n t l y depressed (or even reversed), and the cells are
Published November 20, 1936
256
EFFECTS OF C U R R EN T FLOW.
III
COMPARISONS AND DISCUSSION
Several comparisons have already been made between the behavior
of Nitella and that of Valonia and Halicystis. On the whole, the same
phenomena may be found in each, although the conditions vary considerably. Regular counter E.M.F.'S (polarizations) occur in all, being
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practically non-reactive to currents of the magnitudes ordinarily evoking polarizations (e.g. up to 5 #a/cm.2). The effective resistance
consequently falls nearly (though not quite) to that of dead cells (cf.
footnote 1). However, if sufficiently large currents be passed inward
(e.g., about 10 #a/cm?) polarizations begin to appear, at first with a
very slow rise, an inflection, and then a rapid rise to high positive values
(100 to 200 mv.). Examples are shown in Figs. 9 and 10. This large
P.D. drops off again to low values almost instantly on the cessation
of current flow. Successive inward currents of the same density
produce the effects more rapidly, as do greater densities; they do not,
however, greatly increase the magnitude of total change; indeed, the
higher densities may produce smaller P.D.'s, probably due to injury.
The sharp cusp almost invariably appearing with larger currents is
probably to be explained as a recession due to injury following the
abrupt rise. Return to smaller densities sometimes produces polarizations which were lacking before the larger current flow, causing a
hysteresis much like that in Valonia 3 and Halicystis, 4 but there is
always a point at which polarization again d i s a p p e a r s ~ g a i n a real
threshold effect.
That this striking behavior in the presence of KC1 is not due to
permanent injury is shown by the almost immediate restoration of
normal polarizations, and eventually of stimulations, on re-exposure
of the cells to NaC1 or tap water. On the other hand, polarizations
are entirely lacking, even to very large inward current densities, when
the cells are chloroformed.
Practically no polarizations occur to outward currents in the presence of KC1, even at very high current densities. This is very like
the situation in "variable" Valonia 3 (except that in the latter, polarizations due to temporary "conditioning" by previous inward flow
may occur). In Valonia, however, KC1 does not influence the behavior.
Published November 20, 1936
L. ~. BLINKS
257
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most marked in N#ella, less pronounced in Halicysgs (especially in
impaled cells, where they are very slow, and rather small in magnitude), and almost entirely absent in the variable state of Valonia,
where they appear only on recovery from injury, or by specific treatments. They disappear in Nitella during stimulation, and as long as
recovery is inhibited by KC1; they are suppressed in Valonia by exposure to ammonia in sufficient concentration; while in Halicystis
they persist, or become even greater and faster, during reversal of
P.D. by ammonia, etc. They may be restored in Nitella and Valonia
by the passage of sufficiently large inward currents, which also restore
the positive P.D.
On the other hand, the positive P.D. of both Nitella and ttalizystis
is either destroyed or reversed by the passage of sufficient o u t w a r d
current, with definite threshold effects in each case, and very similar
time curves. Differences occur in recovery, this process in Nitella
being powerful enough to occur in spite of a continued flow of outward
current which had initiated stimulation; while in ttalicystis, recovery
occurs only on cessation or considerable reduction of the original
outward current flow. (In the presence of critical concentrations of
ammonia, however, its P.D. may remain permanently reversed after
cessation of outward current.)
Another common characteristic is the thresholds, which tend to
carry the potentials from one level abruptly to another withou~t intermediate values. These are found not only in the stimulation of
Nitella, the reversal of potential in ttalicystis, and in the production of
positive potentials in Valonia, by current flow; but also in the potential
changes in KCl-treated Nitella, and ammonia-treated ttalicystis.
There is an all-or-none character to these potential levels, about which,
as a base, the more regular counter ~..,~.F.'s build up (when present).
Such levels occur near zero and at 100 my. positive or more in NitelIa;
at some 50-60 Inv. positive and 20-30 Inv. negative in Halicystis; and
at 8 or 10 my. negative and some 50-100 Inv. positive in Valonia.
Very seldom are intermediate levels found; if the potential is carried
by current flow or otherwise to these values, it usually goes beyond
them to the other level, where it is again more stable.
How may these facts be fitted into one picture? The simplest
assumption is that current flow produces two effects; the first, regular
Published November 20, 1936
258
EFFECTS
OF CURRENT
FLOW.
III
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counter E.M.F.'S, dependent upon the presence of some structure (surface film or membrane) but not altering it; the second, a direct alteratire effect upon the structure itself, either destructive or restorative.
For the present, it is unimportant whether the counter E.M.F. is due
to a polarization, dependent on the differential mobility of two species
of ions, or to a static capacity resulting from impermeability (or equal
permeability) to all ions. It is reasonable to suppose, however, that
polarization is involved, since the bioelectric potential itself probably
depends upon a differential mobility of ions, and we have seen that on
the whole counter v..M.F.'s occur when there is also a large, usually
positive P.D. displayed across the protoplasm. Current flows of
moderate density would presumably also produce counter E.~r.F.'S by
setting up across the surface a new concentration gradient of ions
either of those normally responsible for the bioelectric potential itself,
or of any others having appreciable mobility in that surface.
Even up to considerable inward currents, such polarizations are all
that occur, although the counter E.M.F. is finally limited, either by
establishment of a maximum ionic gradient (which would be some
million-fold, to give a potential of 300 to 400 my.), or by "breakdown"
of the surface film. (We might here picture the opening of minute
holes or pores, which promptly repair on cessation of current, since
the injury due even to rather large inward current is not great.) Indeed the contrary holds for we have seen that inward currents have a
strong restorative effect, producing positive P.D. and polarizability
when these are lacking, as in Valonia or KCl-stimulated Nitella.
Outward currents, on the other hand, produce much less counter
E.M.F. before the more drastic changes of stimulation or potential
reversal come into action. Here we must postulate no minor breakdown but a profound alterative effect on the surface film, causing its
temporary disappearance or non-functioning, either with loss of both
P.D. and polarizability in Nitella, or of positive P.D. in Halicystis (where
a second, non-affected surface maintains the negative potential and
continued polarizability). Only in Valonia, where the P.D. is already
negative, and polarizations are either absent or small, does outward
current produce no further effect; we may assume this is because such
Valonia cells are already "permanently stimulated." When polarizability has been temporarily restored by inward currents, however, it
Published November 20, 1936
L. R. BRINKS
259
is destroyed again by outward currents, 3 more rapidly than it disappears spontaneously.
Mechanism of Current Flow Effects
13Bethe, H., Arch. ges. Physiol., 1916, 163, 147.
14Blinks, L. R., Proc. Soc. Exp. Biol. and Med., 1931-32, 9.9, 1186. The results
given here have been recently confirmed in the author's laboratory on still more
favorable material by Mr. R. D. Rhodes, who has obtained objective photographic
and spectrographic evidence of the effects. This is shortly to be arranged for
publication.
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What can cause this destructive action of outwarct currents, and
the restorative effects of inward ones? "Dielectric breakdown" ought
to occur equally well with inward as with outward currents. Differential concentration effects due to ionic movement and changes of
ionic gradients can be expected to occur however. These might
include: (a) hydrogen (or hydroxyl) ions; (b) other inorganic ions,
especially potassium; (c) organic ions of the protoplasm (including
constituents of the surface film itself). These will be discussed in
order.
Hydrogen Ion.--A change of acidity with current flow across membranes or other surfaces has been postulated at least since the
experiments of Bethe, TM who definitely found it to occur with various
models, and believed he demonstrated it in living plant cells. The
latter has been called into question by the present author, who found
that the more obvious of the color effects in living cells containing a
natural indicator, was due to migration of the latter, rather than to a
change of acidity. 14 This does not preclude, however, a change of
acidity in addition, in a layer too thin to be seen, adjacent to the
protoplasm; such is in fact all that might be expected, and all that
would be necessary to produce the effects on the surface itself. Here,
as often, the wrong experiment may have pointed to the right conclusion. For there is much indirect evidence to support the hypothesis
of acidity changes due to current flow. Some of this has been discussed in the papers on Valonia 3 and Halicystis,* where it was shown
that not only by the similarity of their effects, but actually by their
combination in the same experiment, outward currents resembled and
assisted treatments with ammonia and other penetrating bases, while
Published November 20, 1936
260
EFFECTS OF CURRENT FLOW.
IH
15 Blinks, L. R., J. Gen. Physiol., 1933-34, 17, 109.
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inward ones opposed their effects, resembling and assisting treatments
with dilute acids, and certain very active though weakly acidic substances like phenol, cresol, etc. 3 So far, there is not much evidence
of this sort available for Ni~ella, but a similar situation may be
postulated.
There remain two questions concerning the physical and chemical
mechanism of such acidity changes. Is it justified on the basis of
known ionic mobilities? And what effect could acidity changes have
on the surface? Concerning the first, there is not very positive evidence, for pH changes are in most cases rather ineffective in altering
the P.D. Only in the case of Halicystis, and there in only one species, 15
is there evidence of a high mobility of hydrogen ion in the protoplasm.
The general absence of its effect may, however, be due to the necessarily low concentrations of H ion which can be safely applied to cells,
in comparison with the other ionic concentrations present.
Granted that the mobility of H ion might give rise to acidity
changes, it is quite reasonable to postulate effects of such changes on
the surface through, for example, the influence of p H upon the ionization of its constituents, whether these were ampholytes such as proteins, or weak acids such as fatty acids. It has been mentioned before
that the sharply critical all-or-none levels in the cells scarcely coincide
with the long smooth dissociation curves of proteins, which show no
very abrupt and complete change of sign with a small p H shift. More
in keeping with various facts is a lipoid layer, which gives rise to the
observed electrical effects (P.D., polarization) while intact, but ceases
to function when interrupted or removed. A monomolecular arrangement might contribute to the all-or-none nature of its disruption but
this is not necessary, since the effects of a thicker layer would probably
also persist until it was disrupted, the latter occurring by saponification when alkalinity was sufficiently high. That an inner layer, or
the inner side of an outer layer is involved seems indicated by the
action of ammonia which apparently penetrates as NH, across the
outer surface to reach the sensitive region on the inner side, while
other alkalies such as sodium and potassium are less effective, because
Published November 20, 1936
L. ]1. BLINKS
261
te Osterhout, W. J. V., J. Gen. Physiol., 1934-35, 18~ 215.
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they do not penetrate as readily. (When potassium is once admitted,
however, as by stimulation of Nit~tla, it appears to be very tenacious
in its effects.1~)
Restoration of the film might occur by increased acidity, whether
produced by current flow inward, or by metabolic processes. The
latter apparently come spontaneously into prompt and powerful play
immediately after stimulation in Nitella so that recovery occurs in spite
of a continued outward current flow of a density which caused the
original stimulation. They are less powerful in Halicystls, where
recovery occurs only on cessation or diminution of outward current.
And, finally, they are too weak even to maintain the surface, in the
variable state of Valonia, in NiteUa treated with KC1, or in Hallcystis
treated with ammonia, unless assisted in each case by inward current.
Other Inorganic Ions.--Potassium, by virtue of its usual apparently
high mobility in the protoplasm, would presumably be moved from
the sap across the surface by outward currents, to reduce, both P.D.
and resistance when its concentration on the outer side of the surfaces
approached that in the sap. This has been made the basis of a theory
of the action current by Osterhout. 18 There are, however, several
objections to this. (1) Why should stimulation, once initiated, go on
to completion even though current flow ceases (Figs. 3, 4, 5)? (2)
Why should the cells recover from stimulation even though the stimulating current continues to flow, presumably still carrying out potassium ions from the sap across the protoplasm? (3) Why should inward
currents have a restorative effect in the presence of KC1, since here
potassium ions would be brought continuously into contact with the
outer surface? (Similar restoration also occurs in the presence of KC1
with Valonia.)
Nevertheless, it is perfectly clear that potassium produces profound
electrical effects in NiteIta, which are aided and duplicated by outward currents; and are opposed and counteracted by inward currents.
If they are not due to the high mobility of the K ion, then to what?
The author has suggested12 that this may also be through an acidity
effect within the protoplasm, potassium entering the cell either as
Published November 20, 1936
262
~I~ECTS
OF CITRRENT
FLOW.
III
17Jacques, A. G., and Osterhout, W. J. V., Y. Gen. Physiol., 1934-35, 18, 967.
18 Collander, R., abstract in Proc. 6th. Int. BOt. Cong., Amsterdam, 1935, 2, 289.
19Osterhout, W. J. V., and Hill, S. E., J. Gen. Physiol., 1933-34, 17, 87, 99, 105.
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KOH, by formation of an organic salt KX, or by exchange of K for
H ions, but in any case increasing the alkalinity in some critical
region. The surface film is thereby altered or destroyed and recovery
from stimulation is inhibited (unless an inward current is passed).
Too great emphasis is not placed upon this theory, which is supported
mostly by analogy with the ammonia effects in Halicystis15; in view of
recent experiments by Jacques and Osterhout, 17 and by Collander 18 it
may be questioned whether potassium enters Nitella in this manner.
Organic [ons.--Another aspect has been recently given to the
potassium effects by the experiments of Osterhout and HilP ° which
show that stimulation as well as potassium effects can be abolished by
various treatments, such as long exposure to distilled water, and again
restored by various sfibstances. This they explain by the leaching
out of a substance " R " from the cell surface. It is possible that current flow acts upon R, moving it from the cell interior to the surface
during outward current flow, or back from the surface into the interior
during inward flow, depleting it in the surface, and so causing loss of
the potassium effects. Whether R is therefore a cation, and acts by
sensitizing the surface in some way to potassium, remains to be seen.
The simplest hypothesis of all, would be that the surface itself is
composed of an ionic species, capable of migrating in an electric current. If these ions had a negative charge (e.g. were fatty acid radicals) they would be attracted to the outer side of the protoplasm by
the flow of positive current inward, or driven back from the surface
into the protoplasm by outward currents. The observed threshold
might represent the point at which the applied electrical gradient
just overcomes the forces holding the ions in the surface (surface
activity, or chemical affinity for the protoplasmic matrix).
No choice can yet be made between these several hypotheses of the
mechanism of current flow effects, which are suggested for future testing. On the whole, there is the most evidence in favor of a change of
acidity, but this does not preclude other effects, or a combination of
them.
Published November 20, 1936
L. R. BLINKS
263
S~ARY
String g a l v a n o m e t e r records show the effect of current flow u p o n
the bioelectric potential of NiteUa cells. T h r e e classes of effects are
distinguished.
1. Counter ~.,.v.'s, due either to static or polarization capacity,
p r o b a b l y the latter. These account for the high effective resistance
of the cells. T h e y record as s y m m e t r i c a l charge and discharge curves,
which are similar for currents passing inward or o u t w a r d across t h e
p r o t o p l a s m , a n d increase in m a g n i t u d e with increasing current density,
T h e normal positive bioelectric potential m a y be increased b y inward
currents some 100 or 200 m y . , or to a t o t a l of 300 to 400 m y . T h e
regular decrease with o u t w a r d current flow is m u c h less (40 to 50 inv.)
since larger o u t w a r d currents produce the n e x t characteristic effect.
2. Stimulation. This occurs with o u t w a r d currents of a density
which varies s o m e w h a t from cell to cell, b u t is often between 1 a n d 2
# a / e r a . 2 of cell surface. At this threshold a regular counter E.M.r,
2o McClendon, J. F., Am. Y. Physiol., 1912, *$9,302; 1929, 91, 83. Protoplasma,
1929, 7, 561.
~aCf. especially the recent work of Dubuisson, M., Arch. Int. Physiol., 1933,
37, 35; 1934, 38, 85, 460, 468; 1935, 4.1., 177, 511.
22Bozler, E., Y. Cell. and Comp. Physiol., 1935, 6, 217.
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Comparisons with Animal Tissues.--The results with NiteUa are in agreement
with the work of McClendon ~° and others~1who have found a decrease of effective
resistance to accompany the stimulation of muscle. On the other hand Bozler22
has recently found an increase of impedance during the stimulation and contraction
(isometric) of frog sartorius muscle. It is possible that the latter result finds a
parallel in the behavior of Halicystis. In this organism the passage of outward' current produces a reversal of I'.D. bearing much resemblance to the NiteUa
stimulation curve. During the course of reversal, small increments or decrements
of this current have a much larger effect upon the r.D. than they do at either fully
positive or fully negative values.4 The same is true of Valonia during restoration
of the positive potential by inward currents, a Since the changes of I,.D. are always
in the direction of a counter E.~.r. (opposing the flow of current) they Would have
the effect of an increased impedance or effective resistance, and could not be distinguished from such, even though they might be rather due, as here suggested, to
the extreme lability of the cell surface or other structure responsible for the P.D.,
which was being destroyed or reconstituted by the flow of current. I t may be that
the muscle under proper conditions of stimulation shows a similar lability and
sensitivity to current flow, which was reflected in Bozler's measurements.
Published November 20, 1936
26~
EFFECTS OF CURRENT FLOW.
III
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starts to develop but passes over with an inflection into a rapid decrease or even disappearance of positive P.D., in a sigmoid curve with
a cusp near its apex. If the current is stopped early in the Curve
regular depolarization occurs, but if continued a little longer beyond
the first inflection, stimulation goes on to completion even though the
current is then stopped. This is the "action current" or nega'tive
variation which is self propagated down the cell.
During the most profound depression of P.D. in stimulation, current
flow produces.little or no counter ~..~.F., the resistance of the cell being
purely ohmic and very low. Then as the P.D. begins to recover, after
a second or two, counter ~..~.F. also reappears, both becoming nearly
normal in 10 or 15 seconds. The threshold for further stimulation
remains enhanced for some time, successively larger current densities
being needed to stimulate after each action current. The recovery
process is also powerful enough to occur even though the original
stimulating outward current continues to flow during the entire negative variation; recovery is slightly slower in this case however.
Stimulation may be produced at the break of large inward currents,
doubtless by discharge of the enhanced positive P.D. (polarization).
3. Restorative Effects.~The flow of inward current during a negative
variation somewhat speeds up recovery. This effect is still more
strikingly shown in cells exposed to KC1 solutions, which may be
regarded as causing "permanent stimulation" by inhibiting recovery
from a negative variation. Small currents in either direction now
produce no counter ~.M.F., so that the effective resistance of the cells
is very low. With inward currents at a threshold density of some 10
to 20 /za/cm.*, however, there is a counter E.~.F. produced, which
builds up in a sigmoid curve to some 100 to 200 my. positive ~.D.
This usually shows a marked cusp and then fluctuates irregularly
during current flow, falling off abruptly when the current is stopped.
Further increases of current density produce this P.D. more rapidly,
while decreased densities again cease to be effective below a certain
threshold.
The effects in NiteUa are compared with those in Valonia and ttalicystis, which display many of the same phenomena under proper
conditions. It is suggested that the regular counter E.~.F.'S (polarizations) are due to the presence of an intact surface film or other struc-
Published November 20, 1936
~. ~. BLINKS
265
ture offering differential hindrance to ionic passage. Small currents
do not affect this structure, but it is possibly altered or destroyed by
large outward currents, restored by large inward currents. Mechanisms which might accomplish the destruction and restoration are
discussed. These include changes of acidity by differential migration
of H ion (membrane "electrolysis"); movement of inorganic ions such
as potassium; movement of organic ions, (such as Osterhout's substance R), or the radicals (such as fatty acid) of the surface film itself.
Although no decision can be yet made between these, much evidence
indicates that inward currents increase acidity in some critical part
of the protoplasm, while outward ones decrease acidity.
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